A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases catalyze the synthesis of glycoconjugates, including glycolipids, glycoproteins, and polysaccharides, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of the carbohydrate chain. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme (Amado et al. (1999) Biochim Biophys Acta 1473:35-53; Kapitonov et al. (1999) Glycobiology 9:961-78).
Because the glycosylation reaction is highly specific with respect to both the configuration of the sugar residue and the site of the addition, it is expected that unique domain structures for substrate recognition and nucleotide-sugar binding are located within the enzyme molecule. Evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized (Kapitonov et al. (1999) Glycobiology 9:961-78).
Glycosylation is the principal chemical modification to proteins as they pass through Golgi vesicles. Glycosyltransferases of the Golgi do not possess an obvious sequence homology which would suggest a common Golgi retention signal. However, they are all membrane proteins and share type II topology, consisting of an amino terminal cytoplasmic tail, a signal anchor transmembrane domain, a stem region, and a large luminal catalyitc domain. The membrane-spanning domain and its flanking regions contain necessary and sufficient information for Golgi retention of these enzymes (Jaskiewicz (1997) Acta Biochim Pol 44:173-9). ER localized glycosyltransferases can have either a type II topology, like the Golgi glycosyltransferases, or a type I topolgy, e.g., the N-terminus and catalytic domain inside the ER (Kapitonov et al. (1999) Glycobiology 9:961-78). Some glycosyltransferases are present on the cell surface and are thought to function as cell adhesion molecules by binding oligosaccharide substrates on adjacent cell surfaces or in the extracellular matrix. The best studied of these is beta 1,4-galactosyltransferase, which mediates sperm binding to the egg coat and selected cell interactions with the basal lamina (Shur (1993) Curr Opin Cell Biol 5:854-63).
The present invention is based, in part, on the discovery of a novel glycosyltransferase family member, referred to herein as xe2x80x9c47174.xe2x80x9d The nucleotide sequence of a cDNA encoding 47174 is shown in SEQ ID NO:1, and the amino acid sequence of a 47174 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequence of the coding region is depicted in SEQ ID NO:3.
Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 47174 protein or polypeptide, e.g., a biologically active portion of a 47174 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 47174 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3 In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3, wherein the nucleic acid encodes a full length 47174 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs that include a 47174 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 47174 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 47174 nucleic acid molecules and polypeptides. The invention thus also provides vectors and host cells that express the 47174 nucleic acid molecules and polypeptides of the invention. Transgenic animals expressing 47174 nucleic acid molecules and polypeptides of the invention also are provided.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 47174-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 47174 encoding nucleic acid molecule are provided.
In a preferred embodiment, the 47174 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1 or 3. In other embodiments, the 47174 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 2500, 2550, or more contiguous nucleotides of SEQ ID NO:1 or 3. In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 47174 polypeptides.
In another embodiment, the invention provides 47174 polypeptides. Preferred polypeptides are 47174 proteins having a 47174-associated activity, e.g., a glycosyltransferase activity as described herein. In another aspect, the invention features, 47174 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 47174 mediated or related disorders.
In other embodiments, the invention provides 47174 polypeptides, e.g., a 47174 polypeptide having the amino acid sequence shown in SEQ ID NO:2; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, wherein the nucleic acid encodes a full length 47174 protein or an active fragment thereof.
In a preferred embodiment, the 47174 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2. In other embodiments, the 47174 polypeptide is a fragment of at least 15, 20, 50, 100, 150, 200, 300, 400, 500, 600, or more contiguous amino acids of SEQ ID NO:2.
In a related aspect, the invention provides 47174 polypeptides or fragments operatively linked to non-47174 polypeptides to form fusion proteins.
In another aspect, the invention provides methods of screening for agents, e.g., compounds, that modulate the expression or activity of the 47174 polypeptides or nucleic acids, e.g., compounds that modulate neurological activity or function, e.g., central nervous system (CNS) development or function, or that modulate the normal, or aberrant or altered pain response.
In a preferred embodiment, the effect of an agent, e.g., a compound, on the pain response is evaluated by an analgesic test, e.g., the hot plate test, tail flick test, writhing test, paw pressure test, all electric stimulation test, tail withdrawal test, or formalin test.
In a preferred embodiment, the agent, e.g., compound, inhibits 47174 activity.
In a preferred embodiment, the agent, e.g., compound, increases endogenous levels of a 47174 substrate, e.g., glycoconjugates, including glycolipids, glycoproteins, and polysaccharides.
In still another aspect, the invention provides a process for modulating 47174 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant, e.g., decreased or increased expression of the 47174 polypeptides or nucleic acids, such as conditions involving neurological, e.g., CNS, function, e.g., pain response, aberrant or altered pain response, pain related disorders.
In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) an activity of a 47174-expressing cell (e.g., a neural (e.g., a CNS) cell, or a kidney cell), or a response, e.g., a neurological response (e.g., a pain or nociceptive response) in a subject. The method includes contacting the 47174-expressing cell with, or administered to the subject, an agent, e.g., a compound, that modulates the activity or expression of a 47174 polypeptide or nucleic acid, in an amount effective to modulate the activity or the response.
The agent, e.g., the compound, and the 47174-polypeptide or nucleic acid can be contacted in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. The contacting or administering step(s) can be repeated.
In a preferred embodiment, the agent modulates (e.g., increases or decreases) glycosyltransferase activity. In other embodiments, the agent modulates (e.g., increases or decreases) expression of the 47174 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a growth factor, e.g., a neurotrophic or neurotropic factor (e.g., BDNF, NGF, NT-3), a hormone, etc. In those embodiments where cell killing is desired the therapeutic moiety is selected from a cytotoxin, a cytotoxic agent and a radioactive metal ion.
In additional preferred embodiments, the agent is an antisense molecule, a ribozyme, a triple helix molecule, or a 47174 nucleic acid, or any combination thereof.
In a preferred embodiment, the agent is administered in combination with a growth factor, e.g., a neurotrophic or neurotropic factor (e.g., BDNF, NGF, NT-3), a hormone. In other embodiments, the agent is a cytotoxic agent.
In a preferred embodiment, the cell, e.g., the 47174-expressing cell, is a central or peripheral nervous system cell, e.g., a cortical or a hypothalamic cell; a spinal cord cell (e.g., a cell in the DRG); a cell in an area involved in pain control, e.g., a cell in the substantia gelatinosa of the spinal cord, or a cell in the periaqueductal gray matter.
Preferably, the subject is a human, e.g., a patient with a neurological disorder, e.g., a neurodegenerate disorder, a CNS dysfunction, pain or a pain-associated disorder disclosed herein. For example, the subject can be a patient with pain elicited from tissue injury, e.g., inflammation, infection, ischemia; pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches, e.g., migrane; pain associated with surgery; pain related to inflammation, e.g., irritable bowel syndrome; or chest pain. The subject can be a patient with complex regional pain syndrome (CRPS), reflex sympathetic dystrophy (RSD), causalgia, neuralgia, central pain and dysesthesia syndrome, carotidynia, neurogenic pain, refractory cervicobrachial pain syndrome, myofascial pain syndrome, craniomandibular pain dysfunction syndrome, chronic idiopathic pain syndrome, Costen""s pain-dysfunction, acute chest pain syndrome, gynecologic pain syndrome, patellofemoral pain syndrome, anterior knee pain syndrome, recurrent abdominal pain in children, colic, low back pain syndrome, neuropathic pain, phantom pain from amputation, phantom tooth pain, or pain asymbolia. The subject can be a cancer patient, e.g., a patient with brain cancer, bone cancer, or prostate cancer. In other embodiments, the subject is a non-human animal, e.g., an experimental animal, e.g., an arthritic rat model of chronic pain, a chronic constriction injury (CCI) rat model of neuropathic pain, or a rat model of unilateral inflammatory pain by intraplantar injection of Freund""s complete adjuvant (FCA).
In another aspect, the invention features a method of treating or preventing, in a subject, a 47174-associated disorder. The method includes administering to the subject, e.g., a subject at risk of, or afflicted with, a 47174-associated disorder, an agent, e.g., a compound as described herein, that modulates the activity or expression of a 47174 polypeptide or nucleic acid, in an amount effective to treat or prevent the disorder.
In a preferred embodiment, the disorder is neurological, e.g., a neurodegenerative, disorder, a CNS disorder, e.g., a brain or spinal cord related disorder, or a pain or a pain related disorder. In other embodiments, the disorder is a renal disorder.
In a preferred embodiment, the subject is a subject as described herein, e.g., a human, e.g., a patient with a neurological disorder, e.g., a neurodegenerate disorder, or a subject suffering from pain or a pain-associated disorder disclosed herein. In other embodiments, the subject is a patient having a renal disorder.
In still another aspect, the invention features a method for evaluating the efficacy of a treatment of a disorder, e.g., a disorder disclosed herein, in a subject. The method includes treating a subject with a protocol under evaluation; assessing the expression of a 47174 nucleic acid or 47174 polypeptide, such that a change in the level of 47174 nucleic acid or 47174 polypeptide after treatment, relative to the level before treatment, is indicative of the efficacy of the treatment of the disorder.
In a preferred embodiment, the disorder is neurological, e.g., a neurodegenerative, disorder, a CNS disorder, e.g., a brain or spinal cord related disorder, or a pain or a pain related disorder. In other embodiments, the disorder is a renal disorder.
In a preferred embodiment, the subject is a subject as described herein, e.g., a human, e.g., a patient with a neurological disorder, e.g., a neurodegenerate disorder, or a subject suffering from pain or a pain-associated disorder disclosed herein. In other embodiments, the subject is a patient having a renal disorder.
The invention also features a method of diagnosing a disorder, e.g., a disorder disclosed herein, in a subject. The method includes evaluating the expression or activity of a 47174 nucleic acid or a 47174 polypeptide, such that, a difference in the level of 47174 nucleic acid or 47174 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of the disorder.
In a preferred embodiment, the disorder is neurological, e.g., a neurodegenerative, disorder, a CNS disorder, e.g., a brain or spinal cord related disorder, or a pain or a pain related disorder. In other embodiments, the disorder is a renal disorder.
In a preferred embodiment, the subject is a subject as described herein, e.g., a human, e.g., a patient with a neurological disorder, e.g., a neurodegenerate disorder, or a subject suffering from pain or a pain-associated disorder disclosed herein. In other embodiments, the subject is a patient having a renal disorder.
In a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample, is obtained from the subject.
In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 47174 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 47174 nucleic acid or polypeptide.
The invention also provides assays for determining the activity of or the presence or absence of 47174 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 47174 polypeptide or nucleic acid molecule, including for disease diagnosis.
In yet another aspect, the invention features a method for identifying an agent, e.g., a compound, which modulates the activity of a 47174 polypeptide, e.g., a 47174 polypeptide as described herein, or the expression of a 47174 nucleic acid, e.g., a 47174 nucleic acid as described herein, including contacting the 47174 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect of the test compound on the activity of the 47174 polypeptide or nucleic acid to thereby identify a compound which modulates the activity of the 47174 polypeptide or nucleic acid.
In a preferred embodiment, the activity of the 47174 polypeptide is a glycosyltransferase activity, e.g., the synthesis of a glycoconjugate, e.g., a glycolipid, glycoprotein, orolysaccharides.
In a preferred embodiment, the activity of the 47174 polypeptide is modulation of a neural response, e.g., a pain response.
In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.
In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 47174 nucleic acid, or any combination thereof.
In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 47174 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 47174 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 47174 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.